Programmed cell death (PCD) of neurons is very important developmental event to establish functional central nervous system (CNS) via eliminating obsolete neurons. During metamorphic development, certain larval neurons selectively undergo PCD in both vertebrates and invertebrates. Thus, disruption of such developmentally controlled neuronal PCD results in the aberrant formation of adult CNS. Puberty in mammals is comparable to metamorphosis in amphibians and insects, and extensive neuronal modifications in the CNS take place during this period as well. Such a transitional CNS is a causative reason why teens are emotionally and psychologically unstable. However, it is not well understood what types of juvenile neurons are selectively fated to die, and how such selection is made. Drosophila is an excellent model system to tackle these questions, as sophisticated neuro-genetic tools are available. To understand the significance and mechanisms of neuronal PCD during fly metamorphosis, we propose two specific aims in this proposal: (1) we will establish the first comprehensive neuroanatomical map of dying neurons by identifying their neurochemical phenotypes. Since functions of differentiated neurons are dictated by the neurochemical transmitters they bear, this aim will determine which types (functions) of juvenile neurons are programmed to die. Except for 28 larval neurons, we do not know the neurochemical identities of other ~300 doomed neurons. For this aim, we will develop a new in vivo cell death marker to conveniently detect dying neurons. Identification of dying neurons will be done by using specific gal4 drivers. (2) We propose to determine to what extent grim, a critical apoptotic gene in Drosophila CNS, plays a role in the neuronal PCD and to understand how grim expression is regulated. We will employ a novel genome engineering tool, CRISPR, to generate knock-in of grim with reporter genes. These studies will allow us to understand how apoptotic gene expression is induced during metamorphosis. Throughout these works, we expect to train both undergraduate and graduate students and to publish one research paper from each specific aim. Our proposed studies in Drosophila will provide an insight into the role of neuronal PCD during puberty in mammals.